MOL 214

Final Exam – Part A

You must also complete the
Module Exam - Part B

May 21, 2012

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 Exam Code Number:________________

1. Name two functions of the rough endoplasmic reticulum (ER). Cystic fibrosis is caused by a
mutation in the gene that encodes the CFTR protein. How does the CFTR mutation
interfere with processing of CFTR protein by the ER? (3 points)

The rough ER is important for membrane protein synthesis and also is

involved in protein trafficking. The CFTR protein is normally localized in
the cell membrane where it can transport chloride ions outside of the cell.
Failure of CFTR to reach the membrane because of an ER trafficking
defect results in CF.

2. After spending years in the lab, you discover a new antibiotic that is cheap to produce and
effective against every pathogenic bacterium under the sun. Imagine your disappointment
when you realize that this antibiotic is also extremely toxic to humans! It turns out that this
antibiotic prevents myosin from releasing inorganic phosphate. Describe how this affects
muscle contraction. (2 points)

ATP is hydrolyzed to ADP and inorganic phosphate by myosin. Release

of inorganic phosphate triggers the power stroke to generate force. If
inorganic phosphate is never released, the power stroke won’t occur.

3. What are the three basic shapes of bacterial cells? (1.5 points)

Bacilli, cocci, and spirochete.

4. What molecule is used to group organisms into the three domains of life? Why is this
molecule used? (2 points)

rRNA. Ribosomes are found in all living organisms so rRNA sequences

will also be present. Not all genes are so highly conserved.

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5. After sequencing the entire genome of a novel organism by the whole-genome shotgun
approach, you use a computer program to search for start/stop signals for translation and
homology searches based on similarity to cDNAs in the database. You predict
approximately 20,000 genes. Is this an accurate representation of the total number of genes
in the organism? Why or why not? (1.5 points)

No (0.5pts) Looking for start/stop signals for translation and similarity to cDNAs would
only predict protein-coding genes. This would not be an accurate representation of the
total number of genes in the organism because it would miss all the genes encoding
functional RNAs

6. What are two ways that histone modification can result in transcriptional repression? (2
points)

Methylated histones can recruit repressors.

Modified histones can recruit proteins that alter chromatin structure
making it inaccessible to transcriptional machinery.

7. You are trying to identify the factors important for catalyzing RNA splicing in jellyfish. First,
you generate a large quantity of jellyfish cell extracts and test each one for the ability to
splice RNA. When you pretreat the extract with protease, it is still able to catalyze splicing.
When you pretreat the extract with RNase, it no longer catalyzes the splicing reaction.
Based on these observations, what can you conclude about the splicing factor? (2 points)

It is a ribozyme.

8. Why can retroviruses like HIV integrate into the host genome whereas other RNA viruses
like polio, measles, and influenza cannot? (2 points)

Retroviruses can convert RNA to DNA using their reverse transcriptase and this DNA
can be incorporated into the genome. The other viruses don't ever have a DNA
intermediate

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9. You are studying two proteins (P and U) and you suspect that they might interact with each
other. You decide to test your idea by using techniques we discussed in class: affinity
tagging/co-purification and yeast two-hybrid interaction assay. The results from each
experiment are described below.

Affinity purification. You generate two different fusion proteins where an affinity tag is
added to either the N or C terminus of P as pictured below. When you elute the N-
terminal P fusion from the affinity column, you find that you have also purified protein U.
However, when you elute the C-terminal P fusion, you only detect protein P.

Yeast two-hybrid. Regardless of whether P or U is fused to the DNA-binding domain

(BD) or the activation domain (AD) of Gal4, you observe expression of the lacZ reporter
gene. Interestingly, when you express the U-AD construct alone (in the absence of the
P-BD construct), you also detect expression of lacZ. Expression of the U-BD fusion
alone does not activate expression of lacZ.

a) In the affinity purification experiment, why does protein U co-purify with protein P
when the tag is fused to the N-terminus of protein P but not the C-terminus? (2
points)

The C-terminus of P contains the site of interaction with U. Adding a tag

here disrupts the interaction domain. Adding a tag to the N-terminus has
no such effect.

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b) In the two-hybrid experiment, why is expression of the protein U-AD fusion alone
sufficient to activate expression of lacZ? Design an experiment to test your
hypothesis. (3 points)
U is a DNA binding protein that binds to the Gal4 promoter. This positions the
AD to drive expression of lacZ. Note that since the U-BD fusion does not drive
expression of lacZ, U itself is not an activator.

For the experiment, any kind of assay to show that U and DNA co-purify is fine,
but they must mention something about the specific sequence of DNA
recognized by U.

10. After cell division, how is the state of DNA methylation propagated in order to silence genes
epigenetically? (2 points)

After DNA replication, the new strand of DNA, which is unmethylated, is

methylated by a methyltransferase that recognizes hemi-methylated DNA.

11. Why does it make sense that XIST is expressed from the X chromosome that becomes
inactive? (2 points)

Active expression of Xist is required to silence a chromosome. Xist RNA coats the
chromosome, which results in recruitment of chromatin-modifying enzymes that silence
expression of the chromosome

12. A girl develops a neurological defect that doctors trace to a mutation in a paternally
imprinted gene. From which parent did the girl inherit the mutant gene? Explain your
answer. (3 points)
She would have inherited the mutant gene from her mother. Imprinting results in
silencing of the gene so a paternally imprinted gene means that the gene is not
expressed from the chromosome that came from the father and the maternal allele of
the gene is the only allele expressed in the child. Since the father's allele is silent, the
mutant allele must have come from the mother.

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13. You are studying a bacterial protein called Chomper that binds to the disaccharide melibiose
and degrades it into two monosaccharides: glucose and galactose. In order for the
degradation reaction to occur, Chomper must first bind to cAMP. The small molecule
NR81180 inhibits the hydrolysis reaction. You isolate Chomper mutants that are resistant to
NR81180, but the mutations do not map to the Chomper active site. Based on this
information, propose a mechanism for how NR81180 inhibits melibiose degradation. (3
points)

Since NR81180-resistant mutants do not map to the active site of

Chomper, and since cAMP is required for Chomper activity, cAMP
is likely an allosteric activator of Chomper. If so, it is possible that
NR81180 binds to the allosteric site of Chomper and blocks cAMP
binding.

14. Why can you predict the amino acid sequence of a protein from the DNA sequence of the
coding region but you can't necessarily predict the exact DNA sequence of the coding region
of a gene from the amino acid sequence of the protein? (2 points)

The genetic code is degenerate. The same amino acid can be encoded
by more than one codon.

15. Which of the following sugars is found in DNA and which is found in RNA? (1 point)

16. Which of the following bases is a purine and which is a pyrimidine? (1 point)

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17. Pictured below is a DNA replication fork. Indicate the sites of leading and lagging strand
synthesis. (2 points)

18. You have learned about how scientists can create recombinant plasmids containing human
DNA, like DNA that codes for insulin, in bacteria in order to make a lot of protein for
pharmaceutical use. Why is it important to use cDNA rather than genomic DNA in making
these recombinant plasmids? (2 points)

Genomic DNA will likely include introns, which won’t get spliced out in bacteria.

19. When E. coli encounters stress to its cell membrane, expression of the alternative sigma
factor RpoE increases dramatically. What experimental approach could you use to
determine which genes are regulated by RpoE? Is this regulation transcriptional or post-
transcriptional? (3 points)

Use a whole genome microarray to compare between stress and

non-stress conditions. Look for genes that differ between these two
conditions. RpoE is an alternative sigma factor; therefore
regulation is at the level of transcription.

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20. Restriction enzymes are useful for determining the orientation of inserts cloned into
plasmids. Given the following information, draw a map of this hypothetical 8 kb plasmid. (4
points)
• This plasmid can be cut at least once by EcoRI, BamHI, XbaI, and HindIII.
• One of these enzymes cuts twice.
• Restriction digests followed by DNA gel electrophoresis yields bands of the following
sizes:
BamHI = 8 kb
HindIII = 8 kb
BamHI + EcoRI = 2 kb and 4 kb
XbaI + HindIII = 3 kb and 5 kb
XbaI + EcoRI = 1 kb, 3 kb, and 4 kb

21. In class we talked about the mechanism by which expression of the tryptophan biosynthetic
genes is controlled. One crucial feature of this system is the presence of two adjacent
tryptophan codons in trpL leader. What do you predict would happen to expression of the
trp genes if the first codon was mutated to threonine and the second codon was mutated to
alanine? (3 points)

The trp codons serve as a sensor of the amount of intracellular tryptophan available for
incorporation into proteins. When tryptophan is limiting, the ribosome pauses at these
codons allowing formation of the 2-3 stem loop and preventing formation of the 3-4 stem
loop/terminator. The result is expression of the trp genes. Replacement of these
codons would eliminate the tryptophan sensor and thus prevent expression of the trp
genes.

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22. Phosphorylation is one of the most important chemical modifications in biology. Describe
the importance of phosphorylation in the following processes.
a. two component systems (2 points)

Phosphorylation is used to pass information from sensor kinase to

response regulator to generate a transcriptional output.

b. transcriptional elongation (2 points)

CTD must be phosphorylated for RNA pol II to unwind DNA, catalyze NTP
addition, and proofread.

23. Why does translation elongation require GTP hydrolysis? (2 points)

GTP hydrolysis is required for elongation factors to release tRNA and

allow peptide bond formation.
OR
Translocation of ribosome along mRNA requires elongation factor that
hydrolyses GTP.

24. While studying a poorly characterized yeast gene called clueless, you identify a potential
enhancer element close to the transcriptional start site. Design an experiment to determine
whether this element is actually an enhancer, or whether it is part of the clueless promoter.
(3 points)

If this element is an enhancer, it should function independent of its

position. Construct a series of plasmids where the enhancer element is
located at different distances from the predicted promoter. If it is part of
the promoter, moving it will prevent expression of clueless. If it is an
enhancer, it should still be able to enhance clueless transcription when it
is moved away from the transcriptional start site.

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25. You have just identified a new mouse gene called smrtr. To learn more about this gene, you
decide to investigate its expression so you obtain a variety of tissues/organs from mice,
prepare RNA, and perform a northern blot. The blot shows that smrtr is expressed only in
the brain. Your thesis advisor tells you that you should perform an in situ hybridization
experiment to study smrtr further.

a) What additional information would this experiment give you over the information you
obtained from the northern blot? (2 points)

The brain is a complex tissue with lots of cells and the northern blot doesn't tell you
what part of the brain (what cells in the brain) actually express smrtr. The in situ
hybridization experiment would give you more information about where in the brain
smrtr is expressed.

b) Your lab mate cloned another gene called dmbr. The two of you decide to team up to
analyze both smrtr and dmbr in one in situ hybridization experiment. Explain how you
might study both genes at the same time? (2 points)

You would make probes for each, but label the probes differently – the same way you
would do for a microarray experiment.

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Use the following key for questions 26 to 31. The choices may be used once, more than once,
or not at all. However, there is only one correct choice per question. (0.5 points each)

A. initiation phase of translation

B. elongation phase of translation
C. termination phase of translation
D. initiation phase of transcription
E. termination phase of transcription

26. Which of the above involves the ribosome moving three nucleotides at a time along the
mRNA in the 5' to 3' direction?

27. The frequency of which of the above processes is most often regulated by eukaryotic
enhancer elements?

28. Which of the above involves a reaction primarily catalyzed by the RNA component of the
large ribosomal subunit?

29. Which of the above in eukaryotes generally requires the cap structure on an mRNA?

30. Polyadenylation of RNAs in eukaryotes is associated with which of the above steps?
E

31. Which of the above involves the binding of a charged (aminoacyl) tRNA to the A site of the
ribosome?
B

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Multiple choice (1 point each) Choose only one answer

1. Which of the following statements is true?

a) gene expression can be silenced by methylation of histones
b) gene expression is silenced when chromatin is in the open conformation
c) CpG islands block binding by RNA polymerase
d) CpG islands direct mono-allelic gene expression
e) CpG islands are rarely methylated

2. Which of the following is correct?

a) Males have only one X chromosome, so many genes on the X chromosome have to
be expressed at higher levels in males than females.
b) Calico cats are always male.
c) X chromosome gene expression is more similar between males with Klinefelter's
syndrome and unaffected females than between females with Turner's syndrome and
unaffected females
d) Some kids can have both Angelman's and Prader-Willi syndromes

3. The flow of genetic information in cells depends on specific base pairing between
nucleotides. Which of the following correctly matches the type of base pairing with the
process of translation?
a) RNA base-pairs with DNA
b) rRNA base-pairs with mRNA
c) tRNA base-pairs with rRNA
d) tRNA base-pairs with mRNA

4. Researchers investigating a particular gene find that people vary widely in the degree to
which it is expressed. This may be the result of:
a) Variability in DNA sequences that control transcription
b) The gene being on the X chromosome
c) Changes in chromatin structure
d) Whether they live at sea level or at higher elevation
e) All of the above

5. The main limitation of a drug that treats disease by altering chromatin modifications would
be that:
a) The changes would be passed on to daughter cells
b) It would change the number of copies of a gene people inherit
c) The changes would be epigenetic
d) It would change the expression of many other genes
e) All of these

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6. You are analyzing the tissue-specific expression of a poorly characterized gene in mice.
Which type of gene would serve as the best positive control for your experiment?
a) a known gene that exhibits temporally regulated expression
b) a known gene that exhibits spatially regulated expression
c) a known gene that exhibits constitutive expression
d) another poorly characterized or unknown gene

7. Double-stranded RNAs are processed by the enzyme ________ to form small interfering
RNAs (siRNAs). One strand of the siRNA is loaded into a silencing complex named
________. The siRNAs target ________ for ________.
a) RISC, Dicer, mRNA, splicing.
b) Dicer, RISC, mRNA, degradation.
c) Dicer, RISC, rRNA, splicing.
d) RISC, Dicer, rRNA, degradation.

8. Hairpin loops, base-paired stems, and bulges make up what part of RNA structure?
a) primary
b) secondary
c) tertiary
d) quaternary

9. In a beta sheet,
a) adjacent polypeptide strands interact in an extended conformation.
b) beta strands stack at perpendicular angles.
c) R-groups of hydrophobic amino acids stabilize adjacent stretches of amino acid
residues.
d) alpha helices align in parallel or antiparallel arrays.
e) a and b
f) a and c

10. Which of the following statements is true?

a) Some ribozymes cleave themselves.
b) In the RNA world hypothesis, DNA can replicate itself.
c) All enzymes are proteins.
d) RNA viruses can replicate their genomes without proteins.
e) Spliceosomes contain polysomes.

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11. When two strands of DNA from different sources are hybridized in the lab, what provides the
chemical stability for holding the two strands of DNA in a double helix structure?
a) covalent bonds
b) phosphodiester bonds
c) hydrophobic (base-stacking) interactions
d) hydrogen bonds
e) a and b
f) c and d

12. Steps in the base excision repair pathway occur in the following order:
a) Damaged base removal, endonuclease cleavage, nucleotide excision, repair
synthesis ligation.
b) Endonuclease cleavage, damaged base removal, nucleotide excision, repair
synthesis, ligation.
c) Damaged base removal, endonuclease cleavage, ligation, nucleotide excision,
repair synthesis.
d) Endonuclease cleavage, damaged base removal, nucleotide excision, ligation,
repair synthesis.

13. Telomerase directly elongates

a) the 5′ end of the lagging template strand.
b) the 3′ end of the lagging template strand.
c) the 5’ end of the leading template strand.
d) the 3’ end of the leading template strand.
e) a and c.
f) b and d.

14. Which of the statements is not true?

a) Proteins that span the membrane are enriched in hydrophobic side chains
b) The secondary structure of a protein is determined by hydrophobic interactions
c) The size of a protein can be estimated by SDS-PAGE
d) Phosphorylation of a protein alters its charge
e) X-ray crystallography can be used to determine which amino acids in a protein
form an alpha helix

15. The X-ray crystal structure of a eukaryotic release factor protein was solved. Based on your
knowledge of the mechanism by which release factors participate in terminating protein
synthesis, you might expect this protein to most closely resemble which of the following?
a) rRNA from the large ribosomal subunit
b) rRNA from the small ribosomal subunit
c) tRNA
d) small nuclear RNA (snRNA)
e) mRNA

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